1. Field of the Invention
This invention relates to a method for purifying biological conjugates by means of size exclusion chromatography. More particularly, the invention relates to purifying a biological conjugate by means of a chromatography column, whereby there is a high degree of separation between a biological conjugate and an aggregate.
2. Discussion of the Art
An immunoassay is a test that uses complexes comprising antibodies and antigens to generate a detectable and measurable result. A complex comprising and antibody and an antigen is also known as an immuno-complex. Immunoassays are different from other types of laboratory tests, such as colorimetric tests, because most routine clinical chemistry tests utilize chemical reactions between a reagent (a solution of chemicals or other agents) and a biological sample from a patient to generate a test result. For the purpose of an immunoassay, an antibody or an antigen that is able to specifically bind to a target molecule of interest within a biological sample is cross-linked to a label, i.e., a molecule capable of being detected and used for measurement. Examples of a label include a radioactive compound, an enzyme that causes a change of color in a solution, fluorescent markers, a substance that produces light, such as, for example, a chemiluminescent material, and biotin.
Immunoassays utilize one or more selected antibodies or antigens to detect analytes of interest. An analyte is a substance, the concentration of which can be measured by a laboratory test. In immunoassay testing, the analyte can be either an antibody or an antigen. The analytes being measured can be substances that occur naturally in the body, such as, for example, a thyroid hormone). Alternatively, the analytes can be substances that the body produces but that are not typically present in the body, such as, for example, a cancer antigen. Still further, the analytes can be substances that do not naturally occur in the body, such as for example, a drug of abuse. Antibodies possess high specificity and affinity for a specific antigen. It is the specific binding of an antibody to an antigen that allows the detection of analytes by a variety of immunoassay techniques.
The most common format for an immunoassay is the noncompetitive assay format, which generally provides the highest levels of sensitivity and specificity and is typically used for the measurement of critical analytes, such as, for example, cardiac and hepatitis markers. This format is frequently referred to as the “sandwich” assay format, because the analyte is bound (sandwiched) between two highly specific antibody reagents, i.e., the capture antibody and the biological conjugate. A biological conjugate is an entity brought about by the linking of two or more molecules to form a complex that combines properties of its individual components. The reactive site for conjugation can include reactive sites of such molecules as amino acids, peptides, proteins, various sugars, nucleic acids, and oligonucleotides. A wide variety of reaction chemistries for forming biological conjugates can be employed to create such biological conjugates and are well known to those having ordinary skill in the art. Reactive groups that can be used to prepare biological conjugates include amines, thiols, carboxylates, hydroxyl, and aldehydes. In addition to the wide variety of reactive sites and chemical reactions that can be used to form biological conjugates, there is also a wide variety of cross-linking agents that most commonly include zero-length cross-linkers (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), homobifunctinoal crosslinkers (e.g., N-hydroxysuccinimide ester), and heterobifunctional cross-linkers (e.g., succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate succinimide).
The resulting biological conjugate is typically purified after the conjugation reaction. Purification is performed to carried out to remove non-conjugated materials and/or to remove unwanted components, such as, for example, aggregated proteins and aggregated labels, referred to herein as aggregates. The removal of non-conjugated material and large aggregates can greatly improve the performance of an assay; for example, sensitivity can be improved, signal-to-noise ratios can be increased, and wash-cycle efficiency can be improved.
Chromatography is frequently used to purify biological conjugates. Chromatography is a technique wherein the components of a sample, carried by a liquid, are resolved on a stationary phase. While various types of chromatographic processes can be used to purify reaction mixtures containing biological conjugates, such as ion-exchange chromatography, the most common technique is Gel Permeation Chromatography (GPC), also known as size exclusion chromatography. Gel Permeation Chromatography is based on the selective permeation of soluble proteins through a column of particles of a particular size, which particles have pores of a known size. Proteins of a size larger than the pores will not enter the pores. Large proteins that do not enter the pores pass around the particles and are eluted in the void volume (Vo). Very small proteins and salts are retained within the particles until the total permeation volume (Vt) is reached. Proteins that elute between the void volume and the total permeation volume are resolved, based upon the size and shape of their molecules.
It would be desirable to develop a method for purifying a biological conjugate, which method would provide a consistent biological conjugate, consistency being based on size. It would also be desirable to develop a method for purifying a biological conjugate in which the biological conjugate is free from undesired chemical interactions. It would be further desirable to develop a method for purifying a biological conjugate, which method would be capable of resolving both high molecular weight materials and low molecular weight materials from the biological conjugate.